June
3rd
- transformation of 34 BioBricks
4th
- preperation of consumables:
- fresh 50 % glycerol
- new LB plates with cam and with kanamycin (kan)
- 60 glas tubes
- 2 L LB-
- 100 mL steril glas beads for plating
- master plates (6 clones per BioBrick) were made
5th
- colony PCRs on all transformed BioBricks were conducted
- → 2 clones from each master plate were picked
- overnight cultures in freshly prepared 5 mL LB + cam were inoculated
- → 1 culture per BioBrick
6th
- made 2 glycerol stocks of each BioBrick
14th
PCR of E0030 to K1319000
- Q5 and Phusion polymerase are used
| parameter | duration | temp [°C]
|
| denature | 5:00 | 98
|
| denature | 00:30 | 98
|
| anneal | 00:30 | 55
|
| elongate | 00:50 | 72
|
| elongate | 05:00 | 72
|
→ 30 cycles
17th
- colony PCR on following constructs:
| ID | Colonie | Product Length
|
| 1, 2 | K1319000 | 1041
|
| 3, 4 | J23115.E0240 | 1233
|
| 5, 6 | C0062 | 2937
|
| 7, 8 | K516032 | 1219
|
| 9, 10 | J23101.E0240 | 1233
|
| 11 | negativ control | -
|
→ anneling: 50°C
→ elongation: 02:57
20th
- colony PCR of K325108, K325218, C0062
| parameter | duration | temp [°C]
|
| denature | 5:00 | 95
|
| denature | 00:30 | 95
|
| anneal | 00:30 | 49
|
| elongate | 03:15 | 72
|
| elongate | 05:00 | 72
|
→ 30 cycles
- PAGE: 30 min on 1.2 % agarose gel at 110 V
→ weak bands for K325108 a & b at 4.5 kb and 9 kb respectively
23rd
- repetition of yesterdays colony PCR
- preculture for plasmid prep were inoculated
- K1319000 → sequencing
- K592100 → BFP
- S01022 → CFP
- J23101.E0240 → sequencing
- J23115.E0240 → sequencing
- K516132 and J23101.E0240 were plated on LB + cam plates
24th
- 8 plasmid preps were conducted
- overnight cultures of K516132 and J23101.E0240 were inoculated
25th
- preculture for competent NEB10β cells was set up
- samples for sequencing were submitted
- master plates of transformed BioBricks were made
- overnight expression cultures of J23101.E0240 and K516132 were centrifuged and frozen
- fresh 50% glycerol was prepared
26th
- Competent NEB10β cells were made, however, several things went wrong. (For future reference: pre-cool centrifuge, always check if it is indeed spinning, frequently check OD of the culture)
- Colony PCR on the transformed clones looked awful; there were too many cells in the 10 µL reaction volume. Some tubes were not fully sealed during the PCR. Basically, only primers and smear, except for the positive control, which contained a plasmid template instead of cells.
- 2x 500 mL of fresh LB and three sterile flasks were prepared and autoclaved.
27th
| Plasmid | DNA [ng/µl]
|
| J23115.E0240 #1 | 99
|
| J23115.E0240 #2 | 226
|
| K1319000 #6 | 78
|
| K1319000 #1 | 221
|
| K592100 | 240.5
|
| S01022 | 160
|
| J23101.E0240 #5 | 347
|
| J23101.E0240 #6 | 229.5
|
- two cryos stocks of each were prepared
28th
- sequencing
- transformation of 17 BioBricks
30th
|
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